Composition and method for modulating dendritic cell-T cell interaction

ABSTRACT

The present invention relates to the use of a compound that binds to a C-type lectin on the surface of a dendritic cell, in the preparation of a composition for modulating, in particular reducing, the immune response in an animal, in particular a human or another mammal. The composition in particular modulates the interactions between a dendritic cell and a T-cell, more specifically between a C-type lectin on the surface of a dendritic cell and an ICAM receptor on the surface of a T-cell. The compositions can be used for preventing/inhibiting immune responses to specific antigens, for inducing tolerance, for immunotherapy, for immunosuppression, for the treatment of autoimmune diseases, and the treatment of allergy. The compound that binds to a C-type lectin is preferably chosen from mannose, fucose, plant lectins, antibiotics, sugars, proteins or antibodies against C-type lectins. The invention also relates to such antibodies.

The present invention relates to compositions and a method formodulating, in particular increasing or reducing, the immune response inan animal, such as a human or another mammal.

In one embodiment, the invention relates to compositions and a methodfor modulating, and in particular reducing, the adhesion of dendriticcells to T cells.

More specifically, this embodiment of the invention relates tocompositions and a method for modulating, and in particular reducing,the adhesion of C-type lectin receptors on the surface of dendriticcells to the ICAM-receptors on the surface of T cells. By modulatingthis adhesion, both dendritic cell-T cell interactions, such as clusterformation and antigen presentation, as well as for instance primary Tcell responses dependant thereon, can be influenced, resulting in amodulation of the immune response.

The compositions and method of the invention can therefore be used toalter immune responses to specific antigens as well as immune responsescaused by disorders of the immune system, such as may occur inauto-immune diseases or in allergy.

In a further embodiment, the method of the invention can further be usedin the treatment of HIV-infections and similar disorders of the immunesystem, as well as to modulate the immune response to grafts or aftertransplant.

In another embodiment, the invention relates to compounds, compositionsand methods for modulating, and in particular increasing, the immuneresponse in an animal, such as a human or another mammal.

More specifically, in this embodiment, an immune response against aspecific antigen is generated, increased or promoted by presenting saidantigen or an antigenic part thereof to dendritic cells in a form thatcan bind to the C-type lectin receptors on the surface of dendriticcells. The antigen presented in this manner enters the dendritic cell,which in turn presents the antigen to the T-cells, thereby causing animmune response, or an increased immune response, against the antigen.

Further embodiments of the invention relate to prophylactic techniquesas well as diagnostic techniques using the compositions and/or embodyingthe methods as described above.

The invention is based on thee surprising discovery that the adhesion ofdendritic cells to T cells is mediated by a C-type lectin receptor onthe surface of the dendritic cells. It has also been found that thisC-type lectin binds to the ICAM receptors on the surface of T cells.With the term “ICAM receptor(s)” both the ICAM-2 and ICAM-3 receptor aremeant, and in particular the ICAM-3 receptor.

The invention is further based on the finding that the inhibition ofthis C-type lectin receptor on the dendritic cells, such as by knowninhibitors of C-type lectins and/or by specific antibodies directedagainst the C-type lectin receptor, can modulate; and more specificallyreduce, the adhesion of T cells to dendritic cells, and can therebyinfluence the immune response, in particular the initial stages of theimmune response.

WO 96/23882 describes a murine and human receptor with C-type lectinsdomains that is abundantly expressed on the surface of dendritic cellsand thymic epithelial cells. The murine receptor -named “DEC-205”—isdescribed as a 205 kDa protein with an isoelectric point of about 7.5that contains 10 C-type lectin domains and that is homologous to themacrophage mannose receptor (MMR).

WO 96/23882 further describes monoclonal and polyclonal antibodiesagainst DEC-205. However, these antibodies were not able to blockdendritic cell function. In particular, monoclonal and polyclonalanti-DEC-205 antibodies were unable to inhibit the interaction betweendendritic cells and helper T cells, both in vitro (as determined by theinability of anti-DEC-205 to inhibit allogenic T cell proliferation in aone way mixed leucocyte reaction), and in vivo (as determined by theinability of anti-DEC-205 to inhibit an in vivo response, i.e. in alocal graft-versus-host (GVH) reaction). These results suggest that theDEC-205 receptor is not involved in dendritic cell-T cell interaction(i.e. adhesion) and that the anti-DEC-205 antibodies cannot be used tomodulate the immune response.

Curtis et al., in Proc. Natl. Acad. Sci. USA, 89 (1992), p. 8356-8360,aswell as in WO 93/01820, describe a non-CD4 gp120 receptor isolated andcloned from human placenta tissue. This gp120 receptor is expressed onmammalian cells which do not exhibit high levels of CD4, such asplacenta, skeleton muscle, brain, neural and mucosal cells, as well asother tissues and cells including colon, thymus, heart, T cells, B cellsand macrophages (but not in the liver or the kidney). The amino acidsequence of the C-type lectin gp120 receptor disclosed in SEQ IDs no. 1and 2 of WO 93/01820 has a high degree of sequence homology (>98%) withthe C-type lectins that are now found to be present on derndritic cells.

Curtis and WO 93/01820 further discuss the role this C-type lectinreceptor plays in the infection of the aforementioned cells/tissues withHIV, i.e. by binding the major HIV envelope glycoprotein gp120 prior tointernalization of the virion into the cell. It was found thatinhibition of the C-type lectin gp120 receptor can reduce or inhibit HIVinfection of these cells/tissues. As suitable inhibitors, WO 93/01820discloses mannose carbohydrates, fucose carbohydrates, plant lectinssuch as concanavalin A, specific antibiotics such as pradimicin A, andsugars such as N-acetyl-D-glucosaine and galactose (which however aredescribed as less potent). These compounds and compositions containingthem are used either in vitro or in vivo to inhibit the binding of HIVto the cell surface.

WO 93/01820 further discloses that binding of HIV to COS-7 cells can beinhibited by pre-incubation of gp120 with an anti-gp120 monoclonalantibody (named “antibody 110.1”). However, this antibody is notdirected against the C-type lectins, but against the gp120 protein.

However, neither Curtis nor WO 93/01820 mentions or suggests thepresence of such a C-type lectin on dendritic cells, nor do thesereferences mention or suggest their role in dendritic cell—T cellinteraction during the initial stages of an immune response.

WO 95/32734 describes FcγRII (CD32) bridging (or crosslinking)compositions and their use in modulating the immune response to specific30 antigens. This reference is based upon the finding that the bridgingof FcγRII (CD32) molecules on antigen presenting cells (APCs) impairesthe expression of the essentials co-stimulatory molecules B7½ (i.e.prevents their up-regulation) and causes thereby impaires the expressionof (i.e. causes the down-modulation of) the adhesion molecule ICAM-3,with the functional consequence of an impaired capacity of the monocytesto co-stimulate the activation of antigen-specific T cells (i.e.resulting in the modulation of antigen-specific T cellunresponsiveness). The bridging agent is chosen from aggregrated humanIgG molecules or Fc-fragments thereof; bi- or multivalent monoclonalantibodies to FcγRII or fragments thereof, or a fusion of two or morehumane IgG Fc parts.

WO 95/32734 is therefore directed towards modulating (i.e. inhibiting)the co-stimulation -signal required for T cell activation:(i.e., besidesthe primary signal of TcR/CD3 interaction), in particular to induceproliferation and maturation into effector cells. WO95/32734 is notdirected towards modulating dendritic cell—T cell adhesion, nor does itdisclose or suggest either the presence of C-type lectins on (thesurface of) dendritic cells or their interaction with the ICAM-3receptors on T cells.

WO 98/02456 discloses a group II human C-type lectin isolated from astimulated human macrophage library. WO 98/49306discloses a group IVC-type lectin present in human pancreatitis-associated protein (“PAP”).WO 98/41633 discloses a group V human C-type lectin isolated from ahuman tumor clone.

WO 98/02456, WO 98/49306 and WO 98/41633 further disclose methods forproducing antibodies against these C-type lectins.

However, none of these references relates to C-type lectins on dendriticcells; the C-type lectins disclosed in these references differ from theC-type lectins described therein in origin, in biological function, andin structure.

Dendritic cells (DC) are professional antigen-presenting cells thatcapture antigens in the peripheral tissues and migrate via lymph orblood to the T cell area of draining lymph nodes and spleen. Here theypresent processed antigens to naive T cells, initiating antigen-specificprimary T cell responses.

Due to their position in the body surface as immunosurveillance cells,it is likely that DC are the first cells infected with HIV-1 aftermucosal exposure and are therefore implicated to play an important rolein the immunopathogenesis of HIV. It is now generally believed that HIVconverts the normal trafficking process of DC to gain entry into lymphnodes and access to CD4⁺ T cells, as was demonstrated in vivo usingprimary simian immunodeficiency virus infection of macaque as a modelsystem (Spira et al., 1996)(Joag et al., 1997). Productive infection ofDC with HIV-1 has been reported by several investigators(Granelli-Piperno et al., J Virol 72(4), 2733-7, 1998) (Blauvelt et al.,Nat Med 3(12), 1369-75, 1997.) and substantial evidence indicates thatDC pulsed with HIV-1 in vitro can induce a vigorous infection whenco-cultured with T cells (Cameron et. al., Sciences 257(5068), 383-7,1992). Although it is still unclear whether a similar scenario occurs inHIV infected individuals, HIV-1 transmission from DC to T cells couldcontribute to the CD4⁺ T cell depletion observed in AIDS. StudyingHIV-DC interactions should contribute to the understanding of earlyevents of HIV infection and will hopefully lead to strategies aimed atblocking early events in transmission. For a further discussion,reference is also made to WO 95/32734 and WO 96123882.

DC are unique in their ability to interact with and activate resting Tcells. However, prior to the present invention, it was largely unknownhow DC-T cell contact is initiated and regulated. Herein, the role ofICAM-3 in DC-T cell inter-actions is investigated. It is demonstratedthat although DC strongly adhere to ICAM-3, this adhesion is notmediated by LFA-1, αD or any other integrin. In the search for thisnovel ICAM-3 receptor on DC a C-type lectin receptor was cloned,designated DC-SIGN, that is preferentially expressed by DC. Besides itsprominent role in DC-T cell clustering and initiation of T cell responsewe discovered that DC-SIGN is a major HIV-1 receptor involved ininfection of DC and subsequent HIV-1 transmission to T cells. Thus HIV-1and resting T cells exploit a similar highly expressed receptor tointeract with DC.

In a first aspect, the invention relates to the use of a compound thatbinds or can bind to a C-type lectin on the surface of a dendritic cell,in the preparation of a composition for modulating, in particularreducing, the immune response in a animal, in particular a human oranother mammal.

In particular, this aspect of the invention relates to the use of acompound that binds or can bind to a C-type lectin on the surface of adendritic cell, in the preparation of a composition for modulating, inparticular inhibiting, the inter-action(s) between a dendritic cell anda T cell.

More in particular, this aspect of the invention relates to the use of acompound that binds or can bind to a C-type lectin on the surface of adendritic cell in the preparation of a composition for modulating, inparticular reducing, the adhesion between a dendritic cell and a T cell.

Especially, this aspect of the invention relates to the use of acompound that binds or can bind to a C-type lectin on the surface of adendritic cell in the preparation of a composition for modulating, inparticular reducing, the adhesion between a C-type lectin on the surfaceof a dendritic cell and an ICAM receptor on the surface of a T cell.

The amino acid sequence of one C-type lectin that was found to beinvolved in the binding of the dendritic cells to the T-cells is shownin SEQ ID no. 1 and FIG. 9. This C-type lection receptor is essentiallysimilar to the C-type lectin gp120 receptor described by Curtis et al.in Proc. Natl. Acad. Sci. USA, 89 (1992), p. 8356-8360 and in WO93/01820. In particular, it has a high degree of homology (<98%) to theamino acid sequence given in SEQ ID no. 1 of WO 93/01820. It is a groupII C-type lectin of 404 amino acids; with an apparant Mr of about 44kDa; and with a first domain (Met 1 to Ala 76) comprising a cyto-plasmicamino terminus, a second domain (Ile 77 to Val 249) comprising tandemrepeats, and a third domain (Cys 253 to Ala 404) with a high degree ofhomology to other known C-type lectins which are type II membraneproteins. Further characterisation is given below.

In the invention, this C-type lectin on dendritic cells was found tobind to ICAM receptors on the surface of T-cells.

Accordingly, the compositions of the invention can be used to modulate(i.e. alter and/or modify), and more specifically inhibit (i.e. reduceand/or down-tune), the interaction(s) between dendritic cells and Tcells.

Such interactions include the adhesion of T-cells to dendritic cells,for instance in dendritic cell—T cell clustering, T-cell activation andfurther include all interactions that rely on contact between dendriticcells and T-cells, by which is meant either direct cell-to-cell contactor close proximity of dendritic cells and T cells.

Such further interactions includes but are not limited to, processesinvolved in generating an immune response, in particular during theinitial stages of such a response, such as primarysensitation/activation of T-lymphocytes, (i.e. presentation of antigenand/or MHC-bound peptides to T-cells) and co-stimulation of T cells; aswell as processes such as chemical signalling, endocytosis andtrans-epithelial transport. For a discussion of dendritic cell-T cellinteractions in general, all of which may be influenced by thecompositions of the invention, reference is made to the discussion belowas well as to WO 95/32734 and WO 96/23882.

The compositions of the invention can therefore be used to influence theimmunomodulatory ability of dendritic cells; to modulate, and inparticular reduce, dendritic cell-mediated (primary) T cell responses,and/or generally to influence, and in particular inhibit, the immunesystem.

Some specific applications include preventing or inhibiting immuneresponses to specific antigens; inducing tolerance; immunotherapy;immunosuppression, for instance to prevent transplant rejection; thetreatment of auto-immune diseases such as thyroiditis, rheumatoidarthritis, systemic lupus erythematosus (SLE), multiple sclerosis andauto-immune diabetes; and the prevention or treatment of allergies.

The compositions of the invention may also modulate the activation ofother receptors on T cells which are dependant upon the adhesion orclose proximity of dendritic cells to T cells. Furthermore, the findingof the invention that a C-type lectin on dendritic cells binds to theICAM receptors on T cells may open up new strategies or possibilitiesfor influencing the interaction between dendritic cells and T cells, andthereby for modulating the immune system in general.

Furthermore, the compositions of the invention can be used to prevent orreduce the transfer of matter from dendritic cells to T cells, such aschemicals, signalling factors such as chemokines and/or interleukines,etc., and in particular of viral particles such as HIV. In this way, byusing the compositions of the invention, not only can the initialadhesion of HIV to dendritic cells be inhibited, but also the spread ofHIV infection from dendritic cells to T cells.

This finding is of particular importance as it is thought that dendriticcells may serve as a “reservoir” of HIV, in particular during theinitial stages of HIV infection. The compositions of the invention cantherefore not only be used to prevent HIV infection of dendritic cells,but also to reduce the spread of HIV infection to T cells after thedendritic cells have been infected, thereby slowing down the diseaseprocess.

Also, it is known that activation of T cells—i.e. in the lymphglands—plays an important role in the development of AIDS in aHIV-infected patient. It is believed that the compositions of theinvention may be used to prevent, inhibit or at least delay said T-cellactivation and thereby slow the onset and/or the progress of thedisease.

Therefore, in a further aspect, the invention further relates to the useof a compound that binds or can bind to a C-type lectin on the surfaceof a dendritic cell, in the preparation of a composition for inhibitingthe HIV infection of dendritic cells, in particular for inhibiting theadhesion of HIV surface protein (i.e. gp120) to the surface of adendritic cell and thereby the entry of HIV into said dendritic cell.

The invention further relates to the use of a compound that binds or canbind to a C-type lectin on the surface of a dendritic cell, in thepreparation of a composition for inhibiting the transfer of HIV frominfected dendritic cells to non-infected T cells.

Compounds that can be used in the compositions of the invention includeinhibitors for the C-type lectins known per se, including but notlimited to those described in WO 93/01820 as mentioned above.

In general, these are compounds that can bind or adhere to (preferablyin a reversible manner), or that can serve as a ligand for, the C-typelectins, in particular the C-type lectin of SEQ ID no.1/FIG. 9 ornatural variants or equivalents thereof. Examples are mannosecarbohydrates such as mannan and D-mannose; fucose carbohydrates such asL-fucose; plant lectins such as concanavalin A; antibiotics such aspradimicin A; sugars such as N-acetyl-D-glucosamine and galactose (whichhowever are described as less potent); as well as suitablepeptidomimetic compounds and small drug molecules, which can forinstance be identified using phage display techniques. Furthermore,proteins such as gp120 and analogs or fragments thereof or similarproteins with binding capacity to C-type lectins on dendritic cells maybe used, as well as isolated ICAM-receptors and analogs thereof,including part or fragments thereof. Such parts or fragments should thenpreferably still be such that they can bind to the C-type lectins on thesurface of dendritic cells.

However, the use of carbohydrates is usually less desired from atherapeutic point of view, as such they can be rapidly metabolized invivo. Also, the use of plant lectins such as concanavalin A andpradimicin antibiotics can have disadvantages in a therapeutic setting,in particular when treating patients with auto-immune disorders and/orHIV-infections.

Preferably, one or more physiological tolerable and/or pharmaceuticallyacceptable compounds are used, such as defined in WO 93/01820. Forinstance, the use of gp120 or derivatives thereof may cause undesiredside effects, in particular on the nervous system (vide WO 93/01820).

Therefore, according to the invention, preferably an antibody directedagainst a C-type lectins as present/expressed on the surface of adendritic cell, or a part, fragment or epitope thereof, is used. As usedherein, the term antibodies includes inter alia polyclonal, monoclonal,chimeric and single chain antibodies, as well as fragments (Fab, Fv, Fa)and an Fab expression library. Furthermore, “humanized” antibodies maybe used, for instance as described WO 98/49306.

Such antibodies against the C-type lectins of the invention can beobtained as described hereinbelow or in any other manner known per se,such as those described in WO 95/32734, WO 96/23882, WO 98/02456, WO98/41633 and/or WO 98/49306.

For instance, polyclonal antibodies can be obtained by immunizing asuitable host such as a goat, rabbit, sheep, rat, pig or mouse with aC-type lectin or an immunogenic portion, fragment or fusion thereof,optionally with the use of an immunogenic carrier (such as bovine serumalbumin or keyhole limpet hemocyanin) and/or an adjuvant such asFreund's, saponin, ISCOM's, aluminium hydroxide or a similar mineralgel, or keyhole limpet hemocyanin or a similar surface active substance.After an immuneresponse against the C-type lectins has been raised(usually within 1-7 days), the antibodies can be isolated from blood orserum taken from the immunized animal in a manner known per se, whichoptionally may involve a step of screening for an antibody with desiredproperties (i.e. specificity) using known immunoassay techniques, forwhich reference is againt made to for instance WO 96/23882.

Monoclonals may be produced using continuous cell lines in culture,including hybridoma and similar techniques, again essentially asdescribed in the above cited references.

Fab-fragments such as F(ab)₂, Fab′ and Fab fragments may be obtained bydigestion of an antibody with pepsin or another protease, reducingdisulfide-linkages and treatment with papain and a reducing agent,respectively. Fab-expression libraries may for instance be obtained bythe method of Huse et al., 1989, Science 245:1275-1281.

Preferably, a monoclonal antibody against the C-type lectin(s) ondendritic cells is used, more specifically against the peptide with theamino acid sequence shown in/encoded for by SEQ ID no's 1 and 2 and FIG.9, or (an antigenic) part thereof; and such monoclonals are a furtheraspect of the invention. Hereinbelow, the invention will be illustratedby means of two such monoclonals, herein referred to as AZN-D1 andAZN-D2, although similar monoclonals with comparable specificity forC-type lectins may also be used.

In a further aspect, the invention provides a cell line such as ahybridoma that produces antibodies, preferably monoclonal antibodies,against the C-type lectins on dendritic cells, more specifically againstthe peptide with the amino acid sequence shown in/encoded for by SEQ IDno's 1 and 2 and FIG. 9 or (an antigenic) part thereof. Hybridomas thatproduce the abovementioned monoclonals AZN-1 and AZN-2 of the inventionwere deposited on Apr. 8. 1999 with the European Collection of CellCultures under (provisional) ECACC accesion numbers 990400818 and99040819,respectively.

The invention also relates to a method for producing an antibody,preferably a monoclonal antibody, against the C-type lectins ondendritic cells, more specifically against the peptide with the aminoacid sequence shown in (or encoded for) by SEQ ID no's 1 and 2 and FIG.9 or (an antigenic) part thereof, said method comprising cultivating acell or a cell line that produces said antibody and harvesting/isolatingthe antibody from the cell culture.

Neither (monoclonal) antibodies against the C-type lectins on dendriticcells, nor cells or cell lines that produce such antibodies, have todate been described in the art, and it is envisaged that the novelantibodies of the invention will have broad applicability (i.e. besidesthe pharmaceutical/therapeutic uses disclosed herein). Some of theseapplication—which form yet another aspect of the invention—will be clearto the skilled person from the disclosure herein.

For instance, the antibodies of the invention can be used to detect thepresence of (and thereby determine the expression of) C-type lectins inor on tissues or whole cells, as well as the detect the presence ofC-type lectins in other biological samples such as cell fragments or incell preparations. The information thus obtained can then (also) be usedto determine whether the method or compostions of the invention can beapplied to such tissues or cells. The antibodies of the invention couldalso be used to detect (qualitatively and/or quantitatively), isolate,purify and/or produce dendritic cells, for instance in/from biologicalsamples, including biological fluids such as blood, plasma or lymphfluid; tissue samples or cell samples such as bone marrow, skin tissue,tumor tissues, etc; or cell cultures or cultivating media.

For instance, the few methods presently available forisolating/producing dendritic cells from biological samples—such as themethod described in U.S. Pat. No. 5,643,786, comprising leukapheresefollowed by fluorescence-activated cell-sorting—are very cumbersomemulti-step procedures that provide only low yields and heterogenoussamples. As a result, the limited availability of dendritic cells hasseverely hindered research into this important class of cells.

By using the monoclonals of the invention, dendritic cells could beisolated and produced with high(er) yield and with high specificity. Insuch a method, the monoclonals could be used in a manner known per sefor the harvesting, isolation and/or purification of cells frombiological fluids using antibodies.

For instance, the monoclonals could be attached to a column or matrix,to (para)magnetic beads or to a similar solid support, which could thenbe contacted with a biological sample or culture medium containingdendritic cells. The cells that do not attach themselves to the carrierare then separated or removed—e.g. by washing—after which the dendriticcells are separated from the carrier and isolated in a manner known perse.

Also, the monoclonals of the invention could be used to detect/determinethe presence of dendritic cells (and/or C-type lectins) and/or theexpression of genes coding therefor in biological samples, in which theantibodies could again be used in a manner known per se for theanalytical of antibodies, such as competitive inhibition assays orELISA-type immunoassays. For instance, the antibodies could be used incombination with microscopy techniques, cell sorting techniquesincluding flow-cytometry and FACS, techniques based upon solid supportsand/or detectable labels or markers (which can be attached to theantibodies), techniques baed upon (para)magentic beads or any otherdetection or assay technique known per se in which antibodies can beused. Such assays and kits for therein—which besides the antibodies ofthe invention can contain all further components known per se forantibody-based assays, as well as manuals etc.—form a further aspect ofthe invention.

Thus, the monoclonals of the invention constitute a very usefuldiagnostic and research tool, for use both in vitro as well as in vivo.Possible non-limiting fields of application include the study ofdendritic cells and their function and interactions; the study of theimmune system; the detection of dendritic cells and/or C-type lectins incells, tissues or biological fluids such as synovial tissue and skintissue/skin cells (dermal dendritic cells); as well as the study of therole denditric cells play in biological processes or disease mechanisms,such as cancer (as dendritic cells are exploited in vivo in clinicaltrials to irradicate tumor formation and development) and auto-immunediseases (including for instance rheumatoid arthitis).

For a further description of the methods and techniques known per se inwhich the antibodies of the invention can be used, reference is made tothe general textbooks, such as D. P. Sites, A. I. Terr, T. G. Parslow:“Basic and clinical immunology”, 8th Ed., Prentice-Hall (1994); I.Roitt, J. Brostof, D. Male: “Immunology”, 2nd. Ed., ChurchillLivingstone (1994); all incorporated herein by reference. Particularreference is made to the general uses of antibodies and techniquesinvolved therein as mentioned in sections 2.7 to 2.17 of the generalreference work by Janeway-Travers: “Immunobiology, the immune system inhealth and disease”, Third Edition.

A composition of the invention may contain two or more of theabove-mentioned active compounds, or such compounds may be used incombination. For instance, an anti-C-type lectin antibody can beformulated with mannose or fucose carbohydrates, lectins and/orantibiotics such as pridamicin A, whereby a synergistic effect may beobtained.

The compositions of the invention may also contain or be used incombination with known co-inhibitory compounds, such as anti-LF3A; aswell as other active principles known per se, depending upon thecondition to be treated. For instance, the compositions of the inventionmay be formulated or used in combination with immunosuppressants (i.e.for preventing transplant rejection), immunomodulants, antibiotics,auto-antigens or allergens (for instance as described in WO 95/3234 orWO 96/23882), Tumor Necrosis Factor (TNF), and anti-viral agents such asanti-HIV agents and CD4 inhibitors including CD4 directed antibodiessuch as Leu-3A, whereby again a synergistic effect can be obtained.

The compositions of the invention can further be formulated using knowncarriers and/or adjuvantia to provide a pharmaceutical form known perse, such as a tablet, capsule, powder, freeze dried preparation,solution for injection, etc., preferably in a unit dosage form. Suchpharmaceutical forms, their use and administration (single or multidosage form), as well as carriers, excipients, adjuvantia and/orformulants for use therein, are generally known in the art and are forinstance described in WO 93/01820, WO 95/32734, WO 96/23882, WO98/02456, WO 98/41633 and/or WO 98/49306, all incorporated herein byreference. Furthermore, the formulation can be in the form of aliposome, as described in WO 93/01820.

Pharmaceutical formulations of antibodies, their administration and use,are generally described in WO 95/32734, WO 96/23882, WO 98/02456, WO98/41633 and/or WO 98/49306

The compositions of the invention may further be packaged, for instancein vials, bottles, sachets, blisters, etc.; optionally with relevantpatient information leaflets and/or instructions for use.

In a further aspect the invention relates to a method for modulating theimmune response in an animal, in particular a human or another mammal,comprising administering to said animal a compound that binds or canbind to a C-type lectin on the surface of a dendritic cell, preferablyin the form of a composition as described above, in an amount sufficientto alter or modify an immune response.

The compound or composition is in particular administered in such anamount that the interaction(s) between dendritic cells and T cells arealtered or modified, more in particular in such an amount that theadhesion of dendritic cells to T cells is reduced.

This method can be used for preventing and/or treating disorders of theimmune system, as well as to prevent transplant rejection, as describedabove.

The invention further relates to a method for the prevention ortreatment of HIV infections, comprising administering to a HIV infectedpatient or a person at risk of becoming HIV infected, a compound thatcan binds or bind to a C-type lectin on the surface of a dendritic cell,in such an amount that the adhesion of HIV to the dendritic cells, andin particular of the gp120 envelop protein of HIV to the C-type lectinon the surface of dendritic cells, is inhibited.

Also, the invention further relates to a method for the treatment of HIVinfections, comprising administering to a HIV infected patient acompound that binds or can bind to a C-type lectin on the surface of adendritic cell, in such an amount that the transfer of HIV from infecteddendritic cells to non-infected T cells is inhibited.

In a further aspect, the invention is used to modulate, and inparticular generate, increase and/or promote, an immune response in ananimal, such as a human or another mammal, against a specific antigen orcombination of antigens, by presenting said antigen(s) or one or moreantigenic parts thereof to dendritic cells in a form that can bind tothe C-type lectin receptors on the surface of dendritic cells. Theantigen(s) presented in this manner enter(s) the dendritic cell, whichthen in turn presents the antigen to the T-cells, thereby causing animmune response against the antigen(s).

With “a form that can bind to the C-type lectin receptors on the surfaceof dendritic cells” is generally meant that the antigen or antigenicfragment is attached to a compound, ligand or residu that can bind to aC-type lectin on the surface of a dendritic cell, such as thecompounds/ligands mentioned above or a part thereof. Said attachment canfor instance be by (preferably covalent) binding, ligand-ligandinteraction, complexing, ligation, fusion of proteins (e.g. throughexpression of said fusions), or by any other type of physical orchemical interaction or bond that enables the antigen to be presented toa dendritic cell in conjunction with the ligand for the C-type lectin,i.e. combined into a stable or semi-stable entity.

For instance, the antigen can be provided with the abovementionedmannose and fucose carbohydrates as covalently bound groups orside-chains; can be covalently attached to plant lectins such asconcanavalin A or antibiotics such as pradimicin A; or can be providedwith sugar residues such as N-acetyl-D-glucosamine and galactose (whichhowever is less preferred), all of which serve to “direct” the antigento the dendritic cell.

Preferably, the antigen is attached to (e.g. fused with or covalentlybonded to) to a protein that can bind to or serve as a ligand for theC-type lectins, such as gp120 and analogs thereof or the ICAM-receptorsand analogs thereof, or to a part of fragment of such a protein.Alternatively, the antigen can be attached to (e.g. fused with orcovalently bonded to) an antibody directed against the C-type lectins,preferably a monoclonal antibody such as AZN-D1 and AZN-D2 mentionedabove; or to a part or fragment of such an antibody as described above.

The antigen can be any antigen against which an (increased) immuneresponse is to be obtained, or any part or fragment thereof. Preferably,any such part of fragment is such that it per se is capable ofilliciting an immune response, such as an epitope. However, this is notrequired: because according to the invention the fragments are directedto the dendritic cells, i.e. with increased specificity or affinity,part fragments that would normally be incapable of illiciting an immuneresponse may provide an immune response when used in conjunction with aligand for the C-type lectins as described herein. Also, in general,using an antigen in combination with a ligand for the C-type lectins mayincrease the potency of the antigen, i.e. provide a higher or strongerimmune response per unit of antigen administered. In this way,antigens—including those present in serums or vaccines, but alsoretroviral vectors encoding a desired antigen—could be administered at alower dosage and still provide sufficient immune response.

Examples of suitable antigens are cancer antigens including gp 100,g250, p53, MAGE, BAGE, GAGE, MART 1, Tyrosinase related protein II andTyrosinase related protein; all of which can be used to generate animmune response against the tumor cells that contain or express saidantigen. Other types of antigen that can be used in the inventioninclude essentially all antigens used in vaccines against (infectious)diseases, such as influenza, mumps, measles, rubella, diphteria,tetanus, diseases due to infection with micro-organisms such asHaemophilus influenzae (e.g. type b), Neisseria, Bordetella pertussis,Polyomyletus, Influenza virus and Pneumococcus, and generally any otherinfection or disease against which a vaccine can be developed or can beenvisaged, including also parasitical, protozoan and/or viral infectionssuch as HIV and herpes. To provide serums or vaccines, the compounds ofthe invention may further be combined with other antigens known per se.

This aspect of the invention therefore relates to the use of acombination of: 1) a compound that binds to a C-type lectin on thesurface of a dendritic cell; and attached thereto: 2) an antigen or afragment or part thereof; in the preparation of a composition formodulating, in particular generating, increasing and/or promoting, animmune response in a animal, in particular a human or another mammal,against said antigen.

These combinations (e.g. in the form of a complex, a chemical substanceor entity, or a fused protein or protein structure), which as such formanother aspect of the invention, can again be formulated andadministered in a manner known per se, such as described above.

In all the above methods en embodiments, the compounds/compositions usedwill be administered in a therapeutically effective amount, for whichterm reference is generally made to WO 93/01820, WO 95/32734 and/or WO96/23882. The administration can be a single dose, but is preferablypart of a multidose administration regimen carried out over one or moredays, weeks or months.

All terms used herein have the normal meaning in the art, for whichreference can be made to inter alia the definitions given in WO93/01820, WO 95/32734, WO 96/23882, WO 98/02456, WO 98/41633 and/or WO98/49306, analogously applied.

Furthermore, although the invention is described herein with respect tothe specific 44 kDa C-type lectin receptor disclosed herein, it is notexcluded that other, generally similar C-type lectins, including naturalvariants of the sequence of SEQ ID no.1 and FIG. 9, may also be presenton dendritic cells and/or may be involved in dendritic cell—T cellinteraction. Such variants will usually have a high degree of amino acidhomology (more than 80% to more than 90%) with, and/or be functionallyequivalent to the specific C-type lectin disclosed herein. Also, anysuch receptor will generally display properties similar to those asdescribed herein; in particular that inhibition of this receptor, eitherby carbohydrate inhibitors or specific antibodies, will lead to analteration of dendritic cell/T-cell interaction. Any such variantreceptors should however be distinguished from the C-type lectinreceptor disclosed in WO 96/23882, inhibition of which does not resultin inhibition of the interaction of dendritic cells and T-cells.

The invention will now be further illustrated by means of theExperimental Part given hereinbelow, as well as the Figures, in which:

FIGS. 1A-1C are graphs showing: spontaneous adhesion of leukocytes toICAM-1 and ICAM-3 (FIG. 1A); adhesion of leukocytes to ICAM-3 afteractivation of β2-integrins (FIG. 1B); adhesion of DC to ICAM-3 in thepresence of blocking antibodies (20 μg/ml) against β2-integrins(NKI-L19), β1-integrin (AIIB2), ICAM-3. (CBR-IC3/1, CBR-IC3/2) or in thepresence of EDTA (5 mM) or EGTA (5 mM) (FIG. 1C).

FIGS. 2A-2C are graphs showing that the antibodies AZN-D1 and AZN-D2inhibit adhesion of DC to ICAM-3 and recognize an antigen that isspecifically expressed by DC.

FIGS. 3A and 3B show that DC-SIGN is identical to human placenta HIVgp120 binding C-type lectin, as can be seen from SDS-PAGE (FIG. 3A) andby schematic presentation of DC-SIGN isolated from human DC (3B).

FIGS. 4A and 4B show that DC-SIGN, overexpressed in COS7 cells, isrecognized by the anti-DC-SIGN antibody AZN-D1 and binds to ICAM-3.

FIG. 5 shows the tissue distribution of DC-SIGN as determined byimmunohistochemical analysis of the expression of DC-SIGN in tonsils (Aand B) and lymph node sections (C and D) (OM×100).

FIGS. 6A-6D show that DC-SIGN mediated adhesion of DC to ICAM-3 isinvolved in the DC-T-lymphocyte interaction, as demonstrated by DC-SIGNmediated adhesion of DC to ICAM-3 (FIG. 6A); heterotypic cell clusteringof DC with K562-ICAM-3 cells (FIG. 6B); dynamic cell clustering of DCwith resting PBL ( FIG. 6C); and the role of DC-SIGN-ICAM-3 interactionplays in DC-induced T-cell proliferation (FIG. 6D).

FIG. 7 shows that DC SIGN is a receptor for HIV-1 on DC.

FIG. 8 shows that DC SIGN binds to both ICAM-3 as well as ICAM-3expressing K562 cells.

FIG. 9 shows the sequence of DC-SIGN.

EXPERIMENTAL

Dendritic cells (DC) capture antigens and migrate to secondary lymphoidtissues where they present antigens to naive T cells. HIV-1 subvertsthis unique capacity to gain access to CD4⁺ T cells. In the invention, aDC specific C-type lectin was cloned, designated DC-SIGN, that not onlybinds to ICAM-2 and/or ICAM-3 with high affinity but is also able tobind HIV-1. Also, anti-DC-SIGN antibodies were developed that not onlyinhibit transient DC-T cell interactions and DC induced T cellproliferation but also effectively inhibit HBV-1 infection of DC. Thesefindings not only have important consequences for the understanding onCD4-independent HIV entry into DC but also shed new light on the role ofDC-SIGN in initiating primary immune responses.

EXAMPLE 1 Adhesion of DC to ICAM-3 is not Mediated by Integrins

The role of ICAM-3 mediated adhesion in first DC-T cell contact wasinvestigated. Exploiting a novel flow cytrogrometric adhesion assayinvolving ICAM-3-Fc chimera coated fluorescent beads (Geijtenbeek etal.), the capacity of DC, resting peripheral blood lymphocytes (PBL) andmonocytes to bind to this integrin ligand was tested. Immature DC,obtained after culturing of monocytes for 7 days in the presence of IL-4and GM-CSF, strongly bind ICAM-3 without prior activation of β2integrins (72%, FIG. 1A). FIG. 1 demonstrates that the adhesion of DC toICAM-3 is Ca²⁺-dependent and integrin-independent: in FIGS. 1A, B and Cone representative experiment of at least 3 is shown (SD<5%).

1A: Spontaneous adhesion of leukocytes to ICAM-1 and ICAM-3. Freshlyisolated PBL, monocytes and DC were incubated for 30 min. at 37° C. witheither ICAM-1Fc or ICAM-3Fc fluorescent beads. After washing, thepercentage of cells that bound beads was determined by flowcytometry.

1B: Adhesion of lectikocytes to ICAM-3 after activation of β2-integrins.Binding of fluorescent. ICAM-3Fc beads was measured after 30 min. at 37°C. in the presence of either PMA 980 nM) or the activatinganti-β2-integrin antibody KIM185 (10 μg/ml). Inhibition of the LFA-1specific adhesion after PMA activation was determined in the presence ofthe blocking anti-LFA-1 antibody NKI-L15 (20 μg/ml).

1C: Adhesion of DC to ICAM-3 in the presence of blocking antibodies (20μg/ml) against β2-integrins (NKI-L19), β1-integrin (AIIB2), ICAM-3(CBR-IC3/1, CBR-IC3/2) or in the presence of EDTA (5 mM) or EGTA (5 mM).The adhesion was determined as described in FIG. 1A.

This spontaneous binding of DC to ICAM-3 is stronger than that ofmonocytes, whereas resting PBL hardly bind ICAM-3 (FIG. 1A). Adhesion ofDC to ICAM-3 could not be blocked with any anti-αL or anti-β2 integrinantibody (FIG. 1A). In contrast, adhesion of monocytes to ICAM-3 isLFA-1 dependent, since adhesion is blocked by anti-αL antibodies (FIG.1A). Since neither antibodies directed against the other β2 integrinmembers (αDβ2, αMβ2, αXβ2, data not shown), nor antibodies directedagainst other integrins (β1, β7 integrins, FIG. 1B), blocked theadhesion of DC to ICAM-3, it was concluded that the binding of DC toICAM-3 is integrin-independent.

The interaction of DC with ICAM-3-Fc beads is ICAM-3 specific since theanti-ICAM-3 antibodies CBR3/1, CBR3/2 of the invention and a combinationof both antibodies are able to inhibit the adhesion to a large extent(FIG. 1B). Interestingly, adhesion of DC to ICAM-3 could be completelyblocked by EDTA and EGTA (FIG. 1B). These findings strongly suggest thatDC bind ICAM-3 through a Ca²⁺ dependent surface receptor that does notbelong to the β1 or β2 integrin family. This molecule was designated:DC-Specific ICAM-3 Grabbing Non-integrin (DC-SIGN).

EXAMPLE 2 Antibodies Against DC-SIGN Inhibit the DC-ICAM-3 Interaction

To investigate DC-SIGN in more detail, antibodies against the ICAM-3binding receptor were raised. Spleens of mice immunized with DC werefused with SP2/0 myeloma cells and supernatants of the resultinghybridomas were screened for the presence of antibodies capable ofinhibiting DC specific adhesion to ICAM-3. Two hybridomas were selected,cloned and the resulting antibodies were named AZN-D1 and AZN-D2. Bothpurified antibodies strongly inhibit adhesion of DC to ICAM-3, but donot affect LFA-1 mediated adhesion of monocytes to ICAM-3 (FIG. 2A).FIG. 2 demonstrates that antibodies AZN-D1 and AZN-D2 inhibit adhesionof DC to ICAM-3 and recognize an antigen that is specifically expressedby DC:

2A: The monoclonal antibodies AZN-D1 and AZN-D2 (20 μg/ml) blockadhesion of DC but not that of freshly isolated monocytes to fluorescentICAM-3Fc beads. A representative experiment of at least 3 experiments isshown (SD<5%).

2B: DC-SIGN expression increased during DC development. DC were culturedfrom monocytes in the presence of GM-CSF and IL-4. At differenttimepoints the developg DC were analyzed for expression of the monocytemarker CD14, β2 integrin LFA-1 and DC-SIGN. Cells were gated onforward-side scatter and the mean fluorescence is shown in the top rightcorner of the histograms. A representative experiment out of 3 is shown.

2C: DC developing from monocytes, in the presence of GM-CSF and IL-4,increasingly bind to ICAM-3 in a DC-SIGN dependent manner. At differenttime points during culturng cells were harvested and incubated withfluorescent ICAM-3Fc beads in the presence of the blockinganti-β2-integrin antibody AZN-L19 or the AZN-D1 antibody (20 μg/ml).Adhesion was determined as described in FIG. 1A. AZN-D2 inhibitedadhesion to ICAM-3 similar to AZN-D1 (results not shown). Arepresentative experiment out of 3 is shown (SD<5%).

2D: Relative contribution of β2-integrins and DC-SIGN mediated adhesionto ICAM-3 by developing DC. Relative contribution is calculated from theinhibition of adhesion in the presence of AZN-D1 or AZN-L19 as describedin FIG. 2C.

Using AZN-D1 antibodies in flowcytometric analyses it was demonstratedthat DC-SIGN is not expressed by monocytes (FIG. 2B). Cells expressingDC-SIGN can already be detected after 1 day of culture. The expressionlevel of DC-SIGN increases during culture (FIG. 2B). The expression ofthe monocyte marker CD14 gradually decreases during culture and at day 7only a low CD14 expression is observed (FIG. 2B). Further flowcytometricanalyses demonstrated that at day 7 the cells also express high levelsof MHC Class I, II, the β2 integrin p150,95 and CD86 (data not shown),consistent with the differentiation of monocytes into immature DC. Theseresults demonstrate that DC-SIGN is abundantly expressed by DC at day 7,the expression level is several fold higher than that of LFA-1.

Simultaneously, the involvement of DC-SIGN in ICAM-3 binding during thedifferentiation of monocytes into immature DC was investigated (FIG.2C). At onset of the culture (day 0) binding to ICAM-3 by monocytes iscompletely β2 integrin (LFA-1) dependent, as demonstrated by inhibitionof adhesion with the blocking anti-β2 integrin antibody L19 (FIG. 2C).At day 1, when low levels of DC-SIGN are expressed (FIG. 2B), ICAMP-3specific adhesion depends on both β2 integrin (LFA-1) and DC-SIGN (FIG.2C). From day 2 to day 7 the ICAM-3-specific adhesion increases, becomesβ2 integrin-independent (FIG. 2C) and from day 2 is solely mediated byDC-SIGN, since anti-DC-SIGN block the adhesion completely. Maximumadhesion through DC-SIGN is reached at day 7 (FIG. 2C).

Together these results demonstrate that the increase in expression ofDC-SIGN coincides with the observed increase in ICAM-3 binding (FIG. 2Aand B). From these findings it can be concluded that DC-SIGN, recognizedby the anti-bodies AZN-D1 and AZN-D2, is the novel ICAM-3 bindingreceptor expressed by DC.

EXAMPLE 3 DC-SIGN is a 44 kDa Protein

To obtain information regarding the molecular weight of DC-SIGN DC-SIGNwas immunoprecipitated from a lysate of ¹²⁵I-surface labeled DC.Analysis by SDS-PAGE under reducing conditions revealed a single proteinof 44 kDa (FIG. 3A, lanes 1-2). FIG. 3 demonstrates that DC-SIGN isidentical to human placenta HIV gp120 binding C-type lectin:

3A: DC-SIGN is a 44 kDa protein. DC were surface labeled with I¹²⁵,lysed and DC-SIGN was immunoprecipitated with the anti-DC-SIGNantibodies AZN-D1 (lane 1), AZN-D2 (lane 2) and AZN-L19(anti-β2-integrin; lane 3). The immunoprecipitates were analyzed bySDS-PAGE (5-15% gel) followed by autoradiography. The migration of themolecular weight markers is indicated on the left. The arrows indicatethe α-chains of LFA-1 (αL, 180 kDa), MAC-1 (αM, 165 kDa) and p150,95(αX, 150 kDa), the β2 integrin chain (95 kDa) and DC-SIGN (44 kDa).Similar results were obtained in 3 other experiments.

3B: Schematic presentation of DC-SIGN isolated from human DC. The twoboxed peptides (aminoacid positions 296-306 and 187-197 of the humanplacenta gp120 binding C-type lectin ( ) were identified by internalpeptide sequencing of immunoprecipitated DC-SIGn using Edmandegradation. The cDNA encoding DC-SIGN was isolated from DC. The deducedamino acid sequence is 100% identical to that of the human placentagp120 binding C-type lectin ( ). The transmembrane region, the lectindomain and the seven complete and eight partial repeats (R1-R8) areindicated.

Analysis of the immunoprecipitate on a non-reducing SDS-PAGE gel showsthat DC-SIGN exists as a monomer (results not shown). Furthermore, usingICAM-3-Fc coated beads also a 44 kDa protein could be extracted from theDC lysate whereas in the presence of blocking anti-DC-SIGN antibodiesthis protein could not be precipitated with ICAM-3-Fc coated beads(results not shown). These findings demonstrate that DC-SIGN isexpressed by DC as a 44 kDa protein under reducing conditions. Theobservation that ICAM-3 Fc coupled beads only extracted a 44 kDa proteinout of the DC lysate indicates that DC-SIGN has a high affinity forICAM-3, much higher Tan LFA-1 or αDβ2 which are also expressed by DC(FIG. 3A) and have been reported to bind ICAM-3 (Vandervieren et al.,Immunity. 3, 683-690, 1995). Since very low amounts of LFA-1 areimmunoprecipitated in comparison to DC-SIGN (FIG. 3A, lane 1 and 3) thisconfirms that DC-SIGN is more abundantly expressed by DC than LFA-1.Together, these data demonstrates that DC-SIGN is a single polypeptideof 44 kDa and is the primary receptor for ICAM-3 on DC.

EXAMPLE 4 DC-SIGN is Identical to the Human HIV gp120 Binding C-TypeLectin

To identify DC-SIGN a preparative immunoprecipitation from a DC lysatewith the anti-DC-SIGN antibody AZN-D1 and excised the 44 kDa proteinfrom the SDS-PAGE gel was performed. After tryptic digestion, theresulting peptides were extracted from the gel and purified usingpreparative HPLC. Subsequently, the amino acid sequences of 2 peptides(0.5-1 pmol) were determined using the Edman degradation procedure. Bothpeptides consisted of 11 amino acid residues (FIG. 3B) and the peptidesequences were used to screen the EMBL database for homology with knownproteins. Both peptides proved 100% identical to the deduced amino acidsequence of the human HIV gp120-binding C-type lectin (Curtis et al.,1992). This protein has previously been identified exclusively inplacenta as a CD4-independent receptor for the human immuno-deficiencyvirus (HIV) envelope glycoprotein gp120.

RT-PCR analysis with primers based on the gp120-binding C-type lectinsequence, on RNA isolated from DC yielded a PCR product of the expectedlength of 1237 nt. The DC-specific PCR product was cloned and sequencingconfirmed 100% identity with the human gp120-binding lectin (FIG. 3B).Flowcytometric analysis of COS7 cells, transfected with the cDNAencoding the placenta gp120-binding C-type lectin, unequvocally provesthat the gp120 binding C-type lectin is indeed identical to DC-SIGN(FIG. 4A). FIG. 4 demonstrates that DC-SIGN, overexpressed in COS7cells, is recognized by the anti-DC-SIGN antibody AZN-D1 and bindsICAM-3:

4A: AZN-D1 recognizes COS7 cells transfected with the cDNA encodingDC-SIGN (filled) and not mock transfected COS7 cells (open). AZN-D2 gavea similar staining (results not shown).

4B: Adhesion of COS7-DC-SIGN to ICAM-3. COS7 cells were transfected andthe adhesion was determined as described in FIG. 1A, respectively.Adhesion of COS7-DC-SIGN cells to ICAM-3 was measured in the presence ofEGTA (5 mM) and blocking antibodies against DC-SIGN (AZN-D1), ICAM-3(CBR-IC3/1, CBR-IC3/2) and β2 integrinds (AZN-L19). A representativeexperiment out of 3 is shown (SD<5%). About 30% of the transfected COS7cells are stained with anti-DC-SIGN-antibody and therefore expressDC-SIGN. Moreover, the COS7-DC-SIGN cells are able to bind ICAM-3whereas mock transfected COS7 cells are unable to bind ICAM-3 (FIG. 4B).Binding of DC-SIGN expressed by COS7 could be completely inhibited byantibodies against ICAM-3 and DC-SIGN, and was Ca²⁺ dependent since EGTAblocks adhesion completely (FIG. 4B).

It as concluded that the ICAM-3 binding receptor expressed by DC(DC-SIGN) is identical to the placenta HIV gp120 binding C-type lectin(Curtis et al., 1992), a type II transmembrane protein consisting of 404aa with three distinct domains. The N-terminal cytoplasmic domain of 40aa residues is separated by a hydrophobic stretch of 15 aa from a regionwhich consists of seven complete and one incomplete tandem repeat ofnearly identical sequence. The remaining C-terminal region(Cys253-Ala404) shows homology to Ca²⁺-dependent (C-type) lectins (FIG.3B).

EXAMPLE 5 DC SIGN is Specifically Expressed by DC

Flowcytometric analysis of an extensive panel of hematopoietic cellswith the AZN-D1/D2 antibodies demonstrates that the antigen ispreferentially expressed by DC (Table 1). All the hematopoietic cellstested were negative for DC-SIGN expression except for DC. Furthermore,a RT-PCR analysis confirms that the mRNA encoding DC-SIGN isspecifically transcribed in DC which is in accordance with theexpression pattern of the DC-SIGN protein (Table 1).

To further investigate the expression of DC-SIGN in-vivo,immunohistochemical analysis of secondary lymphoid tissues with theanti-DC-SIGN antibodies was performed. These tissues are known tocontain dendritic cells. Sections of tonsils and lymph nodes containedDC-SIGN expressing cells, which were predominantly observed in the Tcell area of both tonsils and lymph nodes (FIG. 5). FIG. 5 shows thetissue distribution of DC-SIGN: Immunohistochemical analysis of theexpression of DC-SIGN in tonsils and lymph node sections (OM×100).Sections were fixed with acetone and the nuclear staining was performedwith Hematein. Staining of DC-SIGN was performed with AZN-D1. Thegerminal center (GC), T-(T) and B-cell (B) areas are depicted.

Consistent with the distribution and morphology of dendritic cells,DC-SIGN expressing cells are not detected in the germinal centres andthe mantle zone of the lymphoid tissues (FIG. 5). Staining of serialsections for CD3 and CD14 confirmed that the DC-SIGN expressing cellsare distinct from T cells and monocytes (data not shown) as was alsodemonstrated by both flowcytometric analysis and RT-PCR of these cells(Table 1).

EXAMPLE 6 DC-SIGN/ICAM-3 Interactions Mediate Transient DC-T LymphocyteClustering

To demonstrate that DC bind to ICAM-3 expressing transfectants in aDC-SIGN dependent manner, the capacity of the leukemic cell line K562transfected with the cDNA encoding ICAM-3 (K562-ICAM-3) to bind to DCwas investigated. As shown in FIG. 6A, DC cluster with K562-ICAM-3 in aDC-SIGN dependent manner, since the interaction can be blocked byanti-DC-SIGN antibodies. No clustering was observed between DC and K562demonstrating that ICAM-3 is the ligand for DC-SIGN. FIG. 6 shows thatDC-SIGN mediated adhesion of DC to ICAM-3 is involved in theDC-T-lymphocyte interaction:

6A: DC-SIGN mediated adhesion of DC to ICAM-3 is dependent on an intactcytoskeleton. Adhesion of DC to ICAM-3 beads was determined with(open-box) or without (filled box) blocking DC-SIGN antibody AZN-D1 inthe presence of Cytpchalasin D, which was titrated in variousconcentration. A representative experiment of 2 experiments is shown(SD<5%).

6B: Heterotypic cell clustering of DC with K562-ICAM-3 cells. K562 andK562 cells stable transfected with the cDNA encoding ICAM-3(K562-ICAM-3) were labeled with the red dye HE (hydroethidine). DC werelabeled with the green dye SFDA. K562 and K562-ICAM-3 were incubatedwith DC (50×10³ cells/cell type) with or without blocking anti-DC-SIGNantibody (AZN-D1; 10 min. pre-incubation) at 37° C. At different timepoints the cells were fixed with paraformaldehyde (0.5%) and theheterotypic cell clustering was measured flow-cytometrically. Arepresentative experiment of 2 experiments is given.

6C: Dynamic cell clustering of DC with resting PbL is mediated byDC-SIGN. DC (50×10³ cells) were pre-incubated with/without theanti-DC-SIGN antibodies AZN-D1 and AZN-D2 (10 μg/ml) for 10 min. at RT.Allogeneic PBL (1×10⁶ cells), labeled with the fluorescent dye Calcein-A(25 μg/10⁷ cells/ml for 30 min. at 37° C.), were added and the cellmixture was incubated at 37° C. The clustering was measured byflow-cytometry. A representative experiment out of 2 is shown.

6D: The DC-SIGN-ICAM-3 interaction is important in DC-inducedT-cell-proliferation. Allogeneic responder T-lymphocytes (100×10³) wereadded to DC-stimulators (1.5×10³) in the presence of blocking antibodies(20 μg/ml) against LFA-3 (TS2/9) and DC-SlGN (AZN-D1, AZN-D2). The cellswere cultured for 4 days. On day 4 the cells were pulsed for 16 h with[³H]methyl-thymidine and the uptake was determined. The results areexpressed as the mean percent of CPM from triplicate wells.

DC-SIGN dependent clustering is transient, with a maximum at 60 minutesindicating that DC-SIGN-ICAM-3 interactions may be actively regulated bythe DC. Furthermore, this phenomenon allows DC to transiently interactwith multiple naive T cells until the interaction is strengthened afterTCR engagement.

To test this it was investigated whether clustering of DC to T cells ismediated by DC-SIGN and whether this interaction is also transient. DCwere incubated with resting allogeneic T cells (DC:T cell, 1:20) Ed theDC-T cell clustering was determined. As shown in FIG. 6B, the clusteringof DC with T cells is transient and reaches a maximum after 20 min(32%). Furthermore, the DC-T cell interaction can be inhibited,(approximately 50%) by anti-DC-SIGN antibodies suggesting that the DC-Tcell clustering is also mediated by other surface receptors. Thus, theDC-T cell clustering is indeed transient and partly mediated byDC-SIGN/ICAM-3 interactions. Similarly, FIG. 8 shows that DC-SIGN bindsnot only with K562 cells expressing cDNA encoding ICAM-3, but also toK562 cells expressing cDNA encoding ICAM-3, and that said binding can beinhibited by both mannan as well as anti DC-SIGN antibodies.

EXAMPLE 7 Proliferation of Resting T Cells Induced by Allogeneic DC isDC-SIGN Dependent

As DC-SIGN binding to ICAM-3 is important for the clustering of DC withT cells, the role of DC-SIGN in DC induced T cell proliferation was alsoinvestigated. Resting T lymphocytes were stimulated with allogeneic DCin the presence or absence of the blocking anti-DC-SIGN antibodies. Asshown in FIG. 6C, the anti-DC-SIGN antibodies AZN-D1 and AZN-D2 bothinhibited the T-lymphocyte proliferation for more than 75%. Similarly,antibodies against the co-stimulatory molecule LFA-3, which binds to CD2on T cells and is also known to mediate T cell proliferation, inhibit Tcell proliferation. A combination of anti-LFA-3 and anti-DC-SIGNantibodies completely inhibits T-cell proliferation (FIG. 6C).

EXAMPLE 8 DC-SIGN is Involved in the HIV-1 Infection of DC

As it was demonstrated hereinabove that DC-SIGN is identical to theplacenta HIV gp120 binding lectin and is abundantly expressed by DC,DC-SIGN might facilitate HIV-1 entry into DC. To investigate this, DCwas pulsed with HIV-1 and productive infection in DC-T cell co-cultureswas measured. DC harvested after 7 days of culture in the presence ofIL-4 and GM-CSF expressed low levels of CD4 (Blauvelt et al., 1997;Granelli-Piperno et al., J Exp Med 184(6), 2433-8, 1996) hand levels ofDC-SIGN (FIG. 7). As shown in FIG. 7 a strong productive infection takesplace when DC are pulsed with HIV-1 for 2 hours, washed and cultured inthe presence of activated PBMC cells. By day 3 of the DC-T cellco-culture the p24 Gag protein, a measure for HIV-1. replication, startsto accumulate in the medium (FIG. 7) demonstrating that HIV-1 isefficiently replicated in the co-culture, similar as has been shown byothers (Blauvelt et. al., 1997; Granelli-Piperno et al., 1998;Granelli-Piperno et al. Curr Biol 9(1), 21-29, 1999). However, when DCprior to the HIV-1 pulse are pre-incubated with anti-DC-SIGN antibodiesand incubated with activated PBMC, HIV-1 replication is inhibited formore than 75%, as shown at day 3 and 5 of DC-T cell co-culture (FIG. 7).When DC were incubated with anti-DC-SIGN antibodies after pulsing withHIV-1, efficient HIV-1 replication was still observed in the DC-T cellco-culture (FIG. 7). These findings demonstrate that anti-DC-SIGNantibodies block HIV-1 infection through inhibition of HIV-1 binding toDC and not the HIV-1 transmission from DC to T cells, indicating thatDC-SIGN act as a major receptor for HIV-1 on DC. Thus, DC-SIGN is highlyexpressed on DC and functions as a DC specific receptor for both ICAM-3and HIV-1.

From the above experimental results, it can inter alia be concluded thatthe initial interaction of DC with T lymphocytes is antigen-independentand transient. This transient nature provides DC with the capacity tointeract with a multitude of T cells until a productive TCR engagementis made. Until now, the mechanism by which this transient process isinitiated has been unclear. Herein, it is demonstrated that theinteraction of a novel DC specific receptor, DC-SIGN, with ICAM-3mediates this transient DC-T cell interaction. DC-SIGN is abundantlyexpressed by DC and it was shown that DC-SIGN serves as a major HIV-1receptor on DC.

An important role for DC during the course of HIV-1 infection is theability to spread HIV-1 to T cells, promoting extensive replication thatleads to the death of CD4+ T cells (Cameron et al., 1992; Cameron: AIDSRes Hum Retroviruses 10(1), 61-71, 1994). Productive HIV-1 infection ofDC has been clearly demonstrated and depends on the development stage ofthe DC (Granelli-Piperno et al., 1998). Immature DC cultured frommonocytes in the presence of IL4 and GM-CSF, are productively infectedby M-tropic HIV-1 strains (Granelli-Piperno et al., 1996;Granelli-Piperno et al., 1998)(Blauvelt et al., 1997) whereas both M-and T-tropic HIV-1 entry into mature DC does not lead to a productiveinfection (Granelli 1998). However, HIV-1 entry into both types of DCdoes lead to an explosive replication upon co-culturing with eitherresting or activated T cells (Granelli 1998, 1999). The initial eventsin HIV-1 infection of target cells include receptor binding and membranefusion. This process is initiated by the high affinity binding of theenvelope glycoprotein gp120 to CD4. However, CD4 alone is not sufficientto initiate fusion, chemokine receptors such as CCR5 and CXCR4 arerequired as co-receptors for the final fusion event to occur (reviewedby Littman et al., 1998)(Dragic et al., Nature 381(6584), 667-73, 1996;Feng et al., Science 272(5263), 872-7, 1996). DC express low amounts ofCD4whereas high levels of DC-SIGN are expressed on the cell surface. Ithas been suggested that productive infection of DC and its ability tocapture and subsequently transmit HIV-1 are mediated through separatepathways. Productive infection of DC is mediated by a CD4-dependentpathway whereas HIV-1 can be captured by DC through a CD4-independentpathway which still enables DC to transmit HIV-1 to T cells (Blauvelt etal. (1997)). Herein, it was shown that DC-SIGN specifically mediatesentry of HIV-1 into DC, as was measured by lack of productive infectionin the DC-T cell co-culture upon preincubation of DC with anti-DC-SIGNantibodies prior to the HIV-1 pulse. Anti-DC-SIGN antibodies do notcompletely inhibit HIV-1 entry into DC. This DC-SIGN-independent pathwayis probably mediated by CD4 ( ) which is expressed at low levels on DC.These results confirm the presence of both a CD4-dependent andindependent pathway for viral entry into DC. Various adhesion moleculeshave been shown to be able to inhibit transmission of HIV-1 from DC to Tcells through interference of DC-T cell contact (Tsunetsugu-Yokota etal., 1997). Anti-DC-SIGN antibodies could not prevent HIV-1 transmissionto T cells when anti-DC-SIGN antibodies were added after the HIV-1 pulseto inhibit the DC-T cell interaction. These data indicate that DC-SIGNserves as a major receptor for HIV-1 entry into DC. The fact that DCexpress high levels of DC-SIGN and low levels of CD4 (FIG. 7) furtherdemonstrates that HIV-1 entry into DC is predominantly mediated byDC-SIGN. The discovery of DC-SIGN as a HIV-1 receptor could be importantin a better understanding of HIV-1 entry into DC. Furthermore, theinhibition of HIV-1 infection observed in the presence of anti-DC-SIGNantibodies will enable the development of anti-DC-SIGN antibodies intherapeutic strategies against viral infection and regional spread ofHIV-1.

DC constitute an heterologous population of cells which are present attrace levels in various tissues. To better define the differentpopulations a lot of effort has gone into the generation of antibodiesthat are directed against DC lineage specific cell surface molecules. Sofar only a few antibodies have been generated which recognize human DCspecific antigens ((Hock et al., Immunol. 83, 573-581, 1994), (deSaint-Vis et al., Immunity 9(3), 325-36, 1998)(Hart et al., 1997).DC-SIGN can now been added to this list of human DC specific antigenssince it was demonstrated herein that at the protein as well as mRNAlevel, of all hematopoietic cells tested, only DC express DC-SIGN (Table1). In situ DC-SIGN is exclusively expressed by DC subsets present inthe T cell area of tonsils and lymph nodes. These mature DC are verypotent in the activation of naive T cells. Therefore, DC-SIGN expressionin situ correlates with its function as an important mediator of DC-Tcell clustering and subsequent T cell activation.

Activation of resting T lymphocytes by antigen presenting cells is acritically important step in the acquired immune response. Located inmost tissues, DC capture and process antigens, and migrate to lymphoidtissues where they interact with and activate naive antigen-specific Tcells. T cells are directed by chemokines to these sites of antigenpresentation. Recently, a DC specific chemokine DC-CK1 was identifiedwhich specifically attracts naive T cells to immune initiation sites(Adema et al., Nature 387, 713-717, 1997). Upon arrival in secondaryIymphoid tissues, T cells interact with D and activation occurs afterTCR recognition of peptides bound to MHC molecules. However, since theaffinity of the TCR for the antigen presented by MRC molecules is verylow and the number of specific MHC-peptide complexes on APC is limited,the interaction of TCR with antigen is usually insufficient to drive theformation of intimate membrane contact between DC and T-lymphocytenecessary for full activation.

To date LFA-1 was the most important receptor for ICAM-3 on DC. However,its role in ICAM-3 binding has now become disputable due to thediscovery herein of DC-SIGN. It was demonstrated that adhesion of DC toICAM-3 is completely mediated by DC-SIGN. DC-SIGN is more abundantlyexpressed by DC than LFA-1 (FIG. 2B). Furthermore, LFA-1 is inactive onDC (FIG. 2C) and its affinity for ICAM-3 is much lower than that ofDC-SIGN for ICAM-3. These data clearly demonstrate that DC-SIGN is theprimary receptor for ICAM-3 on DC. The function for DC-SIGN on DC wasfurther clarified by the finding that anti-DC-SIGN antibodies partiallyinhibited transient DC-T cell clustering. Therefore, DC-SIGN is involvedin the initial DC-T cell interaction in the immune response. A rolewhich was previously attributed to LFA-1. The transient nature of theDC-T cell interaction mediated by DC-SIGN enables DC to interact with alarge number of resting T cells, until a productive TCR mediatedinteraction is made upon which the interaction is stabilized. Theimportance of the DC-SIGN-ICAM-3 interaction is further underscored bythe finding that antibodies against DC-SIGN are able to inhibitallogeneic. DC induced T-lymphocyte proliferation. Moreover, thecombination of antibodies against DC-SIGN and LFA-3, a knownco-stimulatory molecule ( ), almost completely inhibit T-lymphocyteproliferation. Therefore, transient high affinity adhesion of DC-SIGN toICAM-3 plays an important role in the initial antigen-independentinteraction between DC and naive T cells. Presumably, this initial highaffinity interaction enables engagement of the TCR by the antigen boundto the MHC, which subsequently initiates several other adhesiveinteractions between DC and T cells, such as the LFA-1-ICAM-1interaction. Since LFA-1 is inactive on T cells, activation of theTCR/CD3 complex after antigen presentation by DC will result inactivation of LFA-1 and subsequent strong binding of LFA-1 to ICAM-1expressed on DC ( ). Strengthening of the interaction between DC and Tcell via multiple contacts will then lead to full activation of the Tlymphocyte by the DC stimulator ( ).

In conclusion, a novel ICAM-3 -receptor on DC was identified, designatedDC-SIGN, which receptor is specifically expressed by human DC and isinvolved in the initial transient DC-T cell interaction necessary forinitiating an immune response. Interestingly, DC-SIGN is also able tobind the HIV envelope protein gp120 and to facilitate HIV-1 entry intoDC. Various therapeutic and profylactic possibilities and techniques,based upon the findings disclosed herein, will suggest themselves to theskilled person.

EXAMPLE 9 Experimental Procedures

Ex. 9A: Antibodies

The following antibodies were used: KIM185 (anti-β2 integrin, (Andrew etal., Eur. J. Immunol. 23, 2217-2222, 1993), AZN-L19 (anti-β2 integrin,),NKI-L15 (anti-αL, (Keizer et al., Eur. J. Immunol. 15, 1142-1147,1985)), AIIB2 (anti-β1 integrin, (Da Silva et, al., J. Immunol. 143,617-622, 1989)), CBR-IC3/1 and CBR-IC3/2 (anti-ICAM-3 (de Fougerolles etal., J. Exp. Med. 177, 1187-1192, 1993)), CD14 (WT14 ( )), CD4 (wt4 ()). The anti-DC-SIGN antibodies AZN-D1 and AZN-D2 were obtained byimmunizing BALB/c mice with DC and subsequently screening the hybridomasupernatants for the ability to block adhesion of DC to ICAM-3 asmeasured by the fluorescent beads adhesion assay.

Ex. 9B: Cells

DC were cultured from monocytes as described (Sallusto and Lanzavecchia,J. Exp. Med. 179, 1109-1118, 1994; Romani et al., J. Exp. Med. 180,83-93, 1994). Briefly, monocytes were isolated from fresh PBMC by anadherence step. The monocytes were cultured in the presence of IL-4(Schering-Plough, Brussels, Belgium; 500 U/ml) and GM-CSF(Schering-Plough, Brussels, Belgium; 1000 U/ml) for 7 days. At day 4fresh cytokines were added. At day 7 the phenotype of the cultured DCwas confirmed by flowcytometric analysis of the expression of MHC classI and II, CD1a, p150,95 and CD80. Stable K562 transfectants expressingICAM-3 (K562-ICAM-3) were generated by transfection of K562 with 10 μgPCRII ICAM-3 R1 plasmid (gift from Dr D. Simmons) and 2 μg PGK-hygvector (ie Riele et al 1990) by electroporation as described (Lub etal., Mol. Biol Cell 8, 719-728, 1997). Resting T cells (>90% CD3positive) were obtained by centrifugal elutriation of PBMC from bonemarrow of healthy donors, as described (Figdor et al., J. ImmunolMethods 68, 73-87, 1984).

Ex. 9C: Radiolabeling, Immunoprecipitation and Protein SequenceAnalysis.

Cells were surface labeled with Na¹²⁵I (Amersham, Buckinghamshire, UK)through the lactoperoxidase method (Pink and Ziegler, 1979, in: ResearchMethods in Immunology, L. Lefkovits and B. Pernis, eds. (New York:Academic Pres), pp. 169-180.). DC were lysed for 1 hr at 4° C. in lysisbuffer (1% NP-40, 50 mM tri-ethanolamine (pH 7.8), 150 mM NaCl, 1 mnMCaCl₂, 1 MM MgCl₂, 1 mM PMSF, 0.02 mg/ml leupeptin). Nuclear debris wasremoved from the lysate by centrifugation at 13,000 g for 15 min at 4°C. Pre-cleared lysates were incubated for 3 hr with a specific mAbcovalently coupled to Protein A-sepharose CL-4B beads (Pharmacia FineChemicals, Piscataway, N.J.). The immunoprecipitates were extensivelywashed with lysis buffer and analysed by SDS-PAGE according to amodification of the Laemmli procedure (Laemmli, Nature 227, 680-685,1970). Tryptic digestion of the excised protein, purification of theresulting peptides and sequence analysis was performed by EurosequenceBV (Groningen, The Netherlands).

Ex. 9D: Isolation and Expression of the cDNA Encoding DC-SIGN.

Total RNA was isolated by an acidic guanidiniumisothiocyanate-phenol-chloroform procedure (Chomczynski and Sacchi, AnalBiochem 162(1), 156-9, 1987). The cDNA encoding the placenta gp120binding C-type lectin was amplified by RT-PCR on total RNA from DC. PCRprimers were based on the nucleotide sequence of the placenta gp120binding C-type lectin (accession no. M98457, (Curtis et al., 1992)) andthe nucleotide sequences (5′ to 3′) are as follows: XF29,AGAGTGGGGTGACATGAGTG; XR1265, GAAGTTCTGCTACGCAGGAG. The PCR fragment wascloned into the pGEM-T vector (Promega, Madison Wis.) and sequenced. Thenucleotide sequence of the cloned cDNA was identical to that of placentagp120 binding C-type lectin (Curtis et al., 1992). The cDNA wassubsequently cloned into the eukaryotic expression vector pRc/CMV(pRc/CMV-DC-SIGN) and COS7 cells were transient transfected withpRc/CMV-DC-SIGN using the DEAE dextran method (Seed and Aruffo, Proc.Natl. Acad. Sci. U.S.A. 84, 3365-3369, 1987).

Ex. 9EP: Fluorescent Beads Adhesion Assay

Carboxylate-modified TransFluorSpheres (488/645 nm, 1.0 μm; MolecularProbes, Eugene, Oreg.) were coated with ICAM-1 Fc and ICAM-3 Fc asdescribed previously (Geijtenbeek et al., 1999 submitted). Briefly, 20μl streptavidin (5 mg/ml in 50 mM MES-buffer) was added to 50 μlTransFluorSpheres. 30 μl EDAC (1.33 mg/ml) was added and the mixture wasincubated at RT for 2h. The reaction was stopped by the addition ofglycin to a final concentration of 100 mM. The streptavidin-coated beadswere washed three times with PBS (50 mM phosphate, 0.94 NaCl pH 7.4) andresuspended in 150 μl PBS, 0.5% BSA (w/v). The streptavidin-coated beads(15 μl) were incubated with biotinylated goat-anti-human anti-Fc Fab2fragments (6 μg/ml) in 0.5 ml PBA for 2 hours at 37° C. The beads werewashed once with PBS, 0.5% BSA and incubated with human IgG1 Fc fusedligands (ICAM-1 Fc, VCAM-1 Fc; 250 ng/ml) in 0.5 ml overnight at 4° C.The ligand-coated beads were washed, resuspended in 100 μl PBS, 0.5% BSAand stored at 4° C. ICAM-1 Fc and ICAM-3 Fc consist of the extracellularpart of the protein fused to a human IgG1 Fc fragment (provided by Dr D.Simmons). The fluorescent beads adhesion assay was performed asdescribed by Geijtenbeek et al. (submitted). Briefly, cells wereresuspended in Tris-Sodiun-BSA buffer (20 mM Tris-HCl pH 8.0, 150 mMNaCl, 1 mM CaCl₂, 2 mM MgCl₂, 0.5% BSA; 5×10⁶ cells/ml). 50.000 cellswere pre-incubated with/without blocking mAb (20 μg/ml). for 10 min atRT in a 96-wells V-shaped bottom plate. Ligand-coated fluorescent beads(20 beads/cell) and different stimuli/inhibitors were added and thesuspension was incubated for 30 min at 37° C. The cells were washed andresuspended in 100 μl TSA. Adhesion was determined by measuring thepercentage of cells, which have bound fluorescent beads, byflowcytometry using the FACScan (Becton and Dickinson & Co., Oxnard,Calif.).

Ex. 9F: Heterotypic Cell Clustering Assays

Clustering between DC and ICAM-3 expressing cells was assessed byflowcytometry. DC and ICAM-3 expressing cells (2×10⁶ cells/ml) werelabeled respectively with sulfofluorescein (Molecular Probes, Eugene,Oreg.; 50 μg/ml) and hydroethidine (Molecular Probes, Eugene, Oreg.; 40μg/ml) for 1 hour at 37° C. After washing, DC and the ICAM-3 expressingcells were mixed (50×10³ cells each) and incubated at 37° C. Atdifferent time points the cells were fixed with paraformaldehyde (0.5%)and the heterotypic cell clustering was measured by flowcytometry usingthe FACScan (Becton and Dickinson & Co., Oxnard, Calif.).

Clustering between DC with resting T cells was assessed by a differentmethod. DC (50×10³ cells) were pre-incubated with/without theanti-DC-SIGN antibodies AZN-D1 and AZN-D2 (10 g/ml) for 10 min. at RT.Allogeneic PBL (1×10⁶ cells), labeled with the fluorescent dye Calcein-A(Molecular Probes, Eugene, Oreg.; 25 μg/10⁷ cells/ml for 30 min. at37°), were added and the cell mixture was incubated at 37° C. Theclustering was determined by measuring percentage of DC which have boundfluorescent T cells by flowcytometry using the FACScan (Becton andDickinson & Co., Oxnard, Calif.).

Ex. 9G: DC-Induced T Cell Proliferation Assay

Allogeneic responder T-lymphocytes (100×10³) were added toDC-stimulators (1.5×10³) in the presence of blocking antibodies (20_g/ml). The cells were cultured for 4 days. On day 4 the cells werepulsed for 16 h with [³H]methyl-thymidine (1.52 TBq/mmol, 0.5 μCi/well;Amersham, Buckinghamshire, UK) and the uptake was quantified.

Ex. 9H: HIV-1 Infection of DC

HIV-1 _(Ba-L) was grown to high titer in monocyte-derived macrophages(MDM). Seven days after titration of the virus stock on MDM, TCID₅₀ wasdetermined with a p24 antigen ELISA ((Diagnostics Pasteur, Marnes laCoquette, France) and estimated as 10⁴/ml. DC (50×10³), pre-incubatedwith antibodies (50 μg/ml) for 20 min. at RT, were pulsed for 2 h. withwild-type HIV-1 _(Ba-L) (at a multiplicity of infection of 10³infectious units per 10⁵ cells), washed and co-cultured with PHA/IL-2activated PBMC (50×10³). Supernatants were collected 3 and 5 days afterDC-T cell co-culture and p24 antigen levels were measured by a p24antigen ELISA (Diagnostics Pasteur, Marnes la Coquette, France). PBMCwere activated by culturing them in the presence of IL-2 (10 U/ml) andPHA (10 μg/ml).

Ex. 9I: Immunohistochemical Analysis

Cryosections (8 μm) of tonsils and lymph nodes were fixated in 100%aceton (10 min), washed with PBS and incubated with the first antibody(10 μ/ml) for 60 min at RT. After washing, the final staining wasperformed with the ABC-AP Vectastain kit (Vector Laboratories,Burlingame, Calif.) according to the manufacturer's protocol. Nuclearstaining was performed with hematein ( ). TABLE 1 Expression level ofDC-SIGN on hematopoietic cells as determined by flowcytometric analysesand RT-PCR. DC-SIGN DC-SIGN Cell-type expression* mRNA‡ monocytes − − DCday 7 +++ + PBL − − T cells − − B cells − − B-cells (tonsils)$ − n.d.Thymocytes − − Granulocytes − − CD34+ cells − n.d. PBMC (activated#) − −T cell lines† − − monocytic cell − − lines††*mean fluorescence: − = <20, +++ >400 (staining with AZN-D1)‡RT-PCR with the DC-SIGN specific primers XF29 and XR1265 on total RNAisolated from the different cells$isolated from tonsils#activated with PHA (10 μg/ml) and IL-2 (10 U/ml) for 2 days†T cell lines: HSB, PEER, CEM and Jurkat††monocytic cell lines: THP-1, MM6 and U937n.d., not determined

1. A method for increasing an immune response in an animal, comprisingadministering a compound which binds to a C-type lectin on the surfaceof a dendritic cell, with the proviso that the C-type lectin is not theDEC-205 receptor.
 2. The method of claim 1 wherein said animal is amammal.
 3. The method of claim 2 wherein said mammal is a human.
 4. Themethod of claim 1 wherein an antigen is bound to said compound.
 5. Themethod of claim 4 wherein said antigen is bound to said compound by a)covalent binding, b) ligand-ligand interaction, c) complexing, d)ligation, or e) expression of a fusion protein comprising said antigenand said compound.
 6. The method of claim 4 wherein said antigen is acancer antigen.
 7. The method of claim 6 wherein said method generatesan immune response against tumor cells containing or expressing saidcancer antigen.
 8. The method of claim 1 wherein said compound isselected from the group consisting of a mannose carbohydrate, a fucosecarbohydrate, a plant lectin, an antibiotic, a sugar, a protein, and anantibody.
 9. The method of claim 8 wherein said mannose carbohydrate ismannan or D-mannose.
 10. The method of claim 8 wherein said fucosecarbohydrate is L-fucose.
 11. The method of claim 8 wherein said plantlectin is concanavalin A.
 12. The method of claim 8 wherein saidantibiotic is pradimicin A.
 13. The method of claim 8 wherein said sugaris selected from the group consisting of N-acetyl-D-glucosainine andgalactose.
 14. The method of claim 8 wherein said protein is selectedfrom the group consisting of gp120, analogs of gp120 and fragments ofgp120.
 15. The method of claim 1 wherein said C-type lectin is a proteinwith the amino acid sequence of SEQ ID NO:2.
 16. The method of claim 1wherein said C-type lectin is a protein with an amino acid sequence atleast 80% homologous to SEQ ID NO:2.
 17. The method of claim 1 whereinsaid C-type lectin is a protein with an amino acid sequence at least 90%homologous to SEQ ID NO:2.
 18. The method of claim 8 wherein saidantibody is a monoclonal antibody.
 19. The method of claim 8 whereinsaid antibody binds a protein of SEQ ID NO:2.
 20. The method of claim 8wherein said antibody binds a protein with an amino acid sequence atleast 80% homologous to SEQ ID NO:2.
 21. The method of claim 8 whereinsaid antibody binds a protein with an amino acid sequence at least 90%homologous to SEQ ID NO:2.
 22. The method of claim 8 wherein saidantibody is selected from the group consisting of AZN-D1 and AZN-D2. 23.A method for generating an immune response against tumor cells in ananimal, the method comprising administering a compound which binds to aC-type lectin on the surface of the dendritic cell, the compound havingat least a portion of a cancer antigen attached thereto.